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. 2002 Oct 1;367(Pt 1):31–40. doi: 10.1042/BJ20020831

Substrate specificity of the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) assessed by mutagenesis and analysis of synthetic peptides: substrate residues distant from the scissile bond are critical for proteolysis.

Lisbeth S Laursen 1, Michael T Overgaard 1, Claus G Nielsen 1, Henning B Boldt 1, Kathrin H Hopmann 1, Cheryl A Conover 1, Lars Sottrup-Jensen 1, Linda C Giudice 1, Claus Oxvig 1
PMCID: PMC1222882  PMID: 12241545

Abstract

Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and -5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics.

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Selected References

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  1. Bayes-Genis A., Conover C. A., Overgaard M. T., Bailey K. R., Christiansen M., Holmes D. R., Jr, Virmani R., Oxvig C., Schwartz R. S. Pregnancy-associated plasma protein A as a marker of acute coronary syndromes. N Engl J Med. 2001 Oct 4;345(14):1022–1029. doi: 10.1056/NEJMoa003147. [DOI] [PubMed] [Google Scholar]
  2. Bayes-Genis A., Schwartz R. S., Lewis D. A., Overgaard M. T., Christiansen M., Oxvig C., Ashai K., Holmes D. R., Jr, Conover C. A. Insulin-like growth factor binding protein-4 protease produced by smooth muscle cells increases in the coronary artery after angioplasty. Arterioscler Thromb Vasc Biol. 2001 Mar;21(3):335–341. doi: 10.1161/01.atv.21.3.335. [DOI] [PubMed] [Google Scholar]
  3. Bode W. A helping hand for collagenases: the haemopexin-like domain. Structure. 1995 Jun 15;3(6):527–530. doi: 10.1016/s0969-2126(01)00185-x. [DOI] [PubMed] [Google Scholar]
  4. Boldt H. B., Overgaard M. T., Laursen L. S., Weyer K., Sottrup-Jensen L., Oxvig C. Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin. Biochem J. 2001 Sep 1;358(Pt 2):359–367. doi: 10.1042/0264-6021:3580359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bonno M., Oxvig C., Kephart G. M., Wagner J. M., Kristensen T., Sottrup-Jensen L., Gleich G. J. Localization of pregnancy-associated plasma protein-A and colocalization of pregnancy-associated plasma protein-A messenger ribonucleic acid and eosinophil granule major basic protein messenger ribonucleic acid in placenta. Lab Invest. 1994 Oct;71(4):560–566. [PubMed] [Google Scholar]
  6. Byun D., Mohan S., Kim C., Suh K., Yoo M., Lee H., Baylink D. J., Qin X. Studies on human pregnancy-induced insulin-like growth factor (IGF)-binding protein-4 proteases in serum: determination of IGF-II dependency and localization of cleavage site. J Clin Endocrinol Metab. 2000 Jan;85(1):373–381. doi: 10.1210/jcem.85.1.6319. [DOI] [PubMed] [Google Scholar]
  7. Chernausek S. D., Smith C. E., Duffin K. L., Busby W. H., Wright G., Clemmons D. R. Proteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4). Localization of cleavage site to non-homologous region of native IGFBP-4. J Biol Chem. 1995 May 12;270(19):11377–11382. doi: 10.1074/jbc.270.19.11377. [DOI] [PubMed] [Google Scholar]
  8. Conover C. A., Durham S. K., Zapf J., Masiarz F. R., Kiefer M. C. Cleavage analysis of insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis and expression of protease-resistant IGF-binding protein-4 mutants. J Biol Chem. 1995 Mar 3;270(9):4395–4400. doi: 10.1074/jbc.270.9.4395. [DOI] [PubMed] [Google Scholar]
  9. Conover C. A., Faessen G. F., Ilg K. E., Chandrasekher Y. A., Christiansen M., Overgaard M. T., Oxvig C., Giudice L. C. Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum. Endocrinology. 2001 May;142(5):2155–2155. doi: 10.1210/endo.142.5.8286. [DOI] [PubMed] [Google Scholar]
  10. Fowlkes J. L., Serra D. M., Rosenberg C. K., Thrailkill K. M. Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) functions as an IGF-reversible inhibitor of IGFBP-4 proteolysis. J Biol Chem. 1995 Nov 17;270(46):27481–27488. doi: 10.1074/jbc.270.46.27481. [DOI] [PubMed] [Google Scholar]
  11. Fowlkes J. L., Thrailkill K. M., George-Nascimento C., Rosenberg C. K., Serra D. M. Heparin-binding, highly basic regions within the thyroglobulin type-1 repeat of insulin-like growth factor (IGF)-binding proteins (IGFBPs) -3, -5, and -6 inhibit IGFBP-4 degradation. Endocrinology. 1997 Jun;138(6):2280–2285. doi: 10.1210/endo.138.6.5182. [DOI] [PubMed] [Google Scholar]
  12. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  14. Holland D. R., Hausrath A. C., Juers D., Matthews B. W. Structural analysis of zinc substitutions in the active site of thermolysin. Protein Sci. 1995 Oct;4(10):1955–1965. doi: 10.1002/pro.5560041001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holmquist B., Vallee B. L. Metal substitutions and inhibition of thermolysin: spectra of the cobalt enzyme. J Biol Chem. 1974 Jul 25;249(14):4601–4607. [PubMed] [Google Scholar]
  16. Hooper N. M. Families of zinc metalloproteases. FEBS Lett. 1994 Oct 31;354(1):1–6. doi: 10.1016/0014-5793(94)01079-x. [DOI] [PubMed] [Google Scholar]
  17. Hossenlopp P., Seurin D., Segovia-Quinson B., Hardouin S., Binoux M. Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem. 1986 Apr;154(1):138–143. doi: 10.1016/0003-2697(86)90507-5. [DOI] [PubMed] [Google Scholar]
  18. Imai Y., Busby W. H., Jr, Smith C. E., Clarke J. B., Garmong A. J., Horwitz G. D., Rees C., Clemmons D. R. Protease-resistant form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-I actions on porcine smooth muscle cells in culture. J Clin Invest. 1997 Nov 15;100(10):2596–2605. doi: 10.1172/JCI119803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ito A. Mitochondrial processing peptidase: multiple-site recognition of precursor proteins. Biochem Biophys Res Commun. 1999 Nov 30;265(3):611–616. doi: 10.1006/bbrc.1999.1703. [DOI] [PubMed] [Google Scholar]
  20. Jones J. I., Clemmons D. R. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev. 1995 Feb;16(1):3–34. doi: 10.1210/edrv-16-1-3. [DOI] [PubMed] [Google Scholar]
  21. Kitada S., Ito A. Electrostatic recognition of matrix targeting signal by mitochondrial processing peptidase. J Biochem. 2001 Jan;129(1):155–161. doi: 10.1093/oxfordjournals.jbchem.a002827. [DOI] [PubMed] [Google Scholar]
  22. Kitada S., Kojima K., Ito A. Glu(191) and Asp(195) in rat mitochondrial processing peptidase beta subunit are involved in effective cleavage of precursor protein through interaction with the proximal arginine. Biochem Biophys Res Commun. 2001 Sep 28;287(3):594–599. doi: 10.1006/bbrc.2001.5641. [DOI] [PubMed] [Google Scholar]
  23. Kojima K., Kitada S., Ogishima T., Ito A. A proposed common structure of substrates bound to mitochondrial processing peptidase. J Biol Chem. 2000 Oct 12;276(3):2115–2121. doi: 10.1074/jbc.M003111200. [DOI] [PubMed] [Google Scholar]
  24. Kristensen T., Oxvig C., Sand O., Møller N. P., Sottrup-Jensen L. Amino acid sequence of human pregnancy-associated plasma protein-A derived from cloned cDNA. Biochemistry. 1994 Feb 15;33(6):1592–1598. doi: 10.1021/bi00172a040. [DOI] [PubMed] [Google Scholar]
  25. Laursen L. S., Overgaard M. T., Søe R., Boldt H. B., Sottrup-Jensen L., Giudice L. C., Conover C. A., Oxvig C. Pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor binding protein (IGFBP)-5 independent of IGF: implications for the mechanism of IGFBP-4 proteolysis by PAPP-A. FEBS Lett. 2001 Aug 24;504(1-2):36–40. doi: 10.1016/s0014-5793(01)02760-0. [DOI] [PubMed] [Google Scholar]
  26. Lawrence J. B., Oxvig C., Overgaard M. T., Sottrup-Jensen L., Gleich G. J., Hays L. G., Yates J. R., 3rd, Conover C. A. The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):3149–3153. doi: 10.1073/pnas.96.6.3149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mazerbourg S., Overgaard M. T., Oxvig C., Christiansen M., Conover C. A., Laurendeau I., Vidaud M., Tosser-Klopp G., Zapf J., Monget P. Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development. Endocrinology. 2001 Dec;142(12):5243–5253. doi: 10.1210/endo.142.12.8517. [DOI] [PubMed] [Google Scholar]
  28. Mazerbourg S., Zapf J., Bar R. S., Brigstock D. R., Lalou C., Binoux M., Monget P. Insulin-like growth factor binding protein-4 proteolytic degradation in ovine preovulatory follicles: studies of underlying mechanisms. Endocrinology. 1999 Sep;140(9):4175–4184. doi: 10.1210/endo.140.9.6979. [DOI] [PubMed] [Google Scholar]
  29. McQuibban G. A., Butler G. S., Gong J. H., Bendall L., Power C., Clark-Lewis I., Overall C. M. Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1. J Biol Chem. 2001 Sep 24;276(47):43503–43508. doi: 10.1074/jbc.M107736200. [DOI] [PubMed] [Google Scholar]
  30. McQuibban G. A., Gong J. H., Tam E. M., McCulloch C. A., Clark-Lewis I., Overall C. M. Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science. 2000 Aug 18;289(5482):1202–1206. doi: 10.1126/science.289.5482.1202. [DOI] [PubMed] [Google Scholar]
  31. Niidome T., Kitada S., Shimokata K., Ogishima T., Ito A. Arginine residues in the extension peptide are required for cleavage of a precursor by mitochondrial processing peptidase. Demonstration using synthetic peptide as a substrate. J Biol Chem. 1994 Oct 7;269(40):24719–24722. [PubMed] [Google Scholar]
  32. Ogishima T., Niidome T., Shimokata K., Kitada S., Ito A. Analysis of elements in the substrate required for processing by mitochondrial processing peptidase. J Biol Chem. 1995 Dec 22;270(51):30322–30326. doi: 10.1074/jbc.270.51.30322. [DOI] [PubMed] [Google Scholar]
  33. Ou W. J., Kumamoto T., Mihara K., Kitada S., Niidome T., Ito A., Omura T. Structural requirement for recognition of the precursor proteins by the mitochondrial processing peptidase. J Biol Chem. 1994 Oct 7;269(40):24673–24678. [PubMed] [Google Scholar]
  34. Overgaard M. T., Boldt H. B., Laursen L. S., Sottrup-Jensen L., Conover C. A., Oxvig C. Pregnancy-associated plasma protein-A2 (PAPP-A2), a novel insulin-like growth factor-binding protein-5 proteinase. J Biol Chem. 2001 Mar 22;276(24):21849–21853. doi: 10.1074/jbc.M102191200. [DOI] [PubMed] [Google Scholar]
  35. Overgaard M. T., Haaning J., Boldt H. B., Olsen I. M., Laursen L. S., Christiansen M., Gleich G. J., Sottrup-Jensen L., Conover C. A., Oxvig C. Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor. J Biol Chem. 2000 Oct 6;275(40):31128–31133. doi: 10.1074/jbc.M001384200. [DOI] [PubMed] [Google Scholar]
  36. Overgaard M. T., Oxvig C., Christiansen M., Lawrence J. B., Conover C. A., Gleich G. J., Sottrup-Jensen L., Haaning J. Messenger ribonucleic acid levels of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein: expression in human reproductive and nonreproductive tissues. Biol Reprod. 1999 Oct;61(4):1083–1089. doi: 10.1095/biolreprod61.4.1083. [DOI] [PubMed] [Google Scholar]
  37. Oxvig C., Haaning J., Kristensen L., Wagner J. M., Rubin I., Stigbrand T., Gleich G. J., Sottrup-Jensen L. Identification of angiotensinogen and complement C3dg as novel proteins binding the proform of eosinophil major basic protein in human pregnancy serum and plasma. J Biol Chem. 1995 Jun 9;270(23):13645–13651. doi: 10.1074/jbc.270.23.13645. [DOI] [PubMed] [Google Scholar]
  38. Oxvig C., Sand O., Kristensen T., Gleich G. J., Sottrup-Jensen L. Circulating human pregnancy-associated plasma protein-A is disulfide-bridged to the proform of eosinophil major basic protein. J Biol Chem. 1993 Jun 15;268(17):12243–12246. [PubMed] [Google Scholar]
  39. Popken-Harris P., Thomas L., Oxvig C., Sottrup-Jensen L., Kubo H., Klein J. S., Gleich G. J. Biochemical properties, activities, and presence in biologic fluids of eosinophil granule major basic protein. J Allergy Clin Immunol. 1994 Dec;94(6 Pt 2):1282–1289. doi: 10.1016/0091-6749(94)90343-3. [DOI] [PubMed] [Google Scholar]
  40. Qin X., Byun D., Lau K. H., Baylink D. J., Mohan S. Evidence that the interaction between insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-4 is essential for the action of the IGF-II-dependent IGFBP-4 protease. Arch Biochem Biophys. 2000 Jul 15;379(2):209–216. doi: 10.1006/abbi.2000.1872. [DOI] [PubMed] [Google Scholar]
  41. Qin X., Byun D., Strong D. D., Baylink D. J., Mohan S. Studies on the role of human insulin-like growth factor-II (IGF-II)-dependent IGF binding protein (hIGFBP)-4 protease in human osteoblasts using protease-resistant IGFBP-4 analogs. J Bone Miner Res. 1999 Dec;14(12):2079–2088. doi: 10.1359/jbmr.1999.14.12.2079. [DOI] [PubMed] [Google Scholar]
  42. Rawlings N. D., Barrett A. J. Homologues of insulinase, a new superfamily of metalloendopeptidases. Biochem J. 1991 Apr 15;275(Pt 2):389–391. doi: 10.1042/bj2750389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rees C., Clemmons D. R., Horvitz G. D., Clarke J. B., Busby W. H. A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions. Endocrinology. 1998 Oct;139(10):4182–4188. doi: 10.1210/endo.139.10.6266. [DOI] [PubMed] [Google Scholar]
  44. Sottrup-Jensen L. A low-pH reverse-phase high-performance liquid chromatography system for analysis of the phenylthiohydantoins of S-carboxymethylcysteine and S-carboxyamidomethylcysteine. Anal Biochem. 1995 Feb 10;225(1):187–188. doi: 10.1006/abio.1995.1137. [DOI] [PubMed] [Google Scholar]
  45. Sottrup-Jensen L. Determination of halfcystine in proteins as cysteine from reducing hydrolyzates. Biochem Mol Biol Int. 1993 Jul;30(4):789–794. [PubMed] [Google Scholar]
  46. Ständker L., Braulke T., Mark S., Mostafavi H., Meyer M., Höning S., Giménez-Gallego G., Forssmann W. G. Partial IGF affinity of circulating N- and C-terminal fragments of human insulin-like growth factor binding protein-4 (IGFBP-4) and the disulfide bonding pattern of the C-terminal IGFBP-4 domain. Biochemistry. 2000 May 2;39(17):5082–5088. doi: 10.1021/bi992513s. [DOI] [PubMed] [Google Scholar]
  47. Stöcker W., Grams F., Baumann U., Reinemer P., Gomis-Rüth F. X., McKay D. B., Bode W. The metzincins--topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases. Protein Sci. 1995 May;4(5):823–840. doi: 10.1002/pro.5560040502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stöcker W., Zwilling R. Astacin. Methods Enzymol. 1995;248:305–325. doi: 10.1016/0076-6879(95)48021-8. [DOI] [PubMed] [Google Scholar]
  49. Turk B. E., Huang L. L., Piro E. T., Cantley L. C. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat Biotechnol. 2001 Jul;19(7):661–667. doi: 10.1038/90273. [DOI] [PubMed] [Google Scholar]

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